AWWA PATH56544

An epidemiological survey of the distribution of rotavirus types was conducted in Barcelona and Cairo, from June 1998 to December 2001, and from November 1998 to December 1999, respectively. The study was performed with raw sewage samples to evaluate which are the most common rotavirus genotypes in two urban areas with different levels of development. Raw sewage samples from Barcelona (370) collected from June 1998 until December 2001 and from Cairo (35) collected between November 1998 and December 1999 were analyzed. Two different methods were used for viral concentration, lyophilization or adsorption to/elution from positively charged membranes. Fifty ml of Barcelona raw sewage were frozen just after collection and lyophilized with a Bench Top 3 freeze dryer (Virtis) during 48h at -60C, under a pressure of 30-60 mTorr. The pellet was resuspended with 0.5 ml of bidistilled sterile water. Three litres of sewage samples from Cairo were adsorbed on nitro-cellulose membranes, eluted with beef extract-glycine at pH 9.5 and re-concentrated by organic flocculation to obtain a final volume of 1 ml. In this way, viruses were 100 and 3,000 times concentrated, respectively. Viral RNA was extracted from concentrated samples by the Boom's method (Boom et al, 1990) and stored at -80C until further processed. An RT-PCR-Southern blot hybridization method based on the amplification of a fragment from the VP6 coding segment was used as a general screening procedure for rotavirus detection. Positive samples were typed by previously described methods based on the VP7 or VP4 coding segments (Gouvea et al, 1990; Gentsch et al, 1992). Both methods consist of a first round RT-PCR with generic primers followed by a multiplex seminested PCR with type-specific primers. The primers used in each of both reactions allows us to detect the main human G-types (G1, G2, G3 and G4) and the additional G5 type, and the P1A, P1B, P2 and P3 types. Includes 9 references.

Product Details

Edition:

Vol. - No.

Published:

09/22/2002

Number of Pages:

4

File Size:

1 file , 120 KB